Americans spend at least $100 billion each year on low back pain, a ubiquitous job-related disability and a leading contributor to missed days of work. While the etiology of disc disease is multifactorial, there is general agreement that loss of water and a decrease in osmotic pressure compromises cellular function and exacerbates the disease state. We have recently shown that T1? MRI and Opening Pressure (OP), parameters of disc hydration and osmotic status, provide quantitative measures of both tissue degeneration and back pain. Despite recognition of these facts, molecular mechanisms that control the disc water content and osmotic pressure remain poorly understood. We have shown that TonEBP, the only known osmoresponsive transcription factor is expressed in the nucleus pulposus (NP) and is diminished in the disease state. Based on these findings, we propose to test an innovative overarching hypothesis: TonEBP activity is linked to osmotic status of the NP and correlates with T1? and OP and controls critical NP survival and functional activities; compromised TonEBP function promotes development of disease. To test this central hypothesis, first, we will measure the expression of TonEBP and its target genes in human degenerate discs in relationship to measures of hydration status and pain: T1? and OP. We will then define the importance of TonEBP by suppressing its expression and evaluating its effects on NP cell apoptosis, autophagy and senescence. Second, we will determine the mechanism by which TonEBP controls expression of water transporters (aquaporins) and proteoglycans, molecules required for the maintenance of the osmotic properties of the disc. In addition, we will measure expression of key enzymes required for glycan synthesis and sulfation. We will determine if the disease process has altered the ability of TonEBP to respond to osmotic shifts. We will ascertain if restoration of TonEBP activity in diseased human cells using angiotensin II (ANG II), PDGF-BB or TonEBP expression plasmid stimulates matrix synthesis and aquaporin levels. The third goal of the investigation is to determine if inactivation of TonEBP compromises osmotic and biomechanical properties of the disc in vivo. Using lentiviral-ShTonEBP injection in rat discs and conditional inactivation of TonEBP in TonEBPf/f mice, we will determine if suppression causes alterations in T1? signal, matrix composition and biomechanical properties of the disc. Finally, using a well characterized rat model of disc disease, we will attempt to restore TonEBP expression/activity using either PDGF-BB or ANG II and monitor changes. Outcomes from these studies will provide foundation for innovative strategies aimed at restoring osmotic and water balance mediated through TonEBP, thereby delaying the development or progression of degenerative disc disease in the aging spine.